title: Expression, purification and biological characterization of human vasostatin120-180 in Pichia pastoris.
abstract: Angiogenesis is a major feature of tumor growth and metastasis. As such, targeting the tumor neovasculature is an attractive strategy for effective cancer therapy. Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. Our laboratory previously reported a strategy for expression and purification of human vasostatin120-180 (VAS) in a GST-tagged fusion form using Escherichia coli expression system. However, the yield of 7.2 mg per liter of culture was relatively low and the protein activity was also limited. In this study, the biologically active and soluble VAS was cloned and expressed in Pichia pastoris. The yield of the active VAS was about 20 mg/L of the P. pastoris culture medium. The recombinant protein was purified to homogeneity, and confirmed to be biologically active. The recombinant VAS could efficiently inhibit angiogenesis and endothelial cell proliferation in vitro. Moreover, the P. pastoris-derived VAS showed relatively higher protein activity than E. coli-derived VAS. Furthermore, it can inhibit in vivo xenograft tumor growth and prolong the tumor doubling time significantly by inhibiting angiogenesis. Taken together, this is the first report on the heterologous expression of VAS in P. pastoris, and P. pastoris is a highly efficient and cost-effective expression system for large amount production of biologically active recombinant VAS for potential therapeutic application.
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, Jiangsu, PR China.
authors.forename: Yuli
authors.initials: Y
authors.lastname: Lin
authors.forename: Xuguang
authors.initials: X
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Lu
authors.forename: Hongqin
authors.initials: H
authors.lastname: Zhuang
authors.forename: Zi-Chun
authors.initials: ZC
authors.lastname: Hua
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D037282:Calreticulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D037282:Calreticulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; C121986:vasostatin
keywords: Anti-angiogenesis; Pichia pastoris; Recombinant protein production; Tumor therapy; Vasostatin120–180